Two kinds of effector mechanisms mediate immune responses. Some immune responses are mediated by specific molecules, called antibodies, that are carried in the blood and lymph. The synthesis of antibodies occurs in a subset of lymphocytes called B lymphocytes or B cells. Antibody-mediated immunity is called humoral immunity. Other immune responses are mediated by cells. All the leukocytes (white cells) of the blood participate in cell-mediated immunity (CMI). However, the specificity of the response depends upon a subset of lymphocytes called T lymphocytes or T Cells. Most immune responses involve the activity and interplay of both the humoral and the cell-mediated branches of the immune system.
Ultraviolet radiation is a major environmental carcinogen and the primary cause of non-melanoma skin cancer worldwide (Urbach, 1978). In addition to being a carcinogen, UV radiation is also immunosuppressive, and studies using mice have demonstrated an intimate link between the immunosuppressive effects of UV radiation and the development of skin cancer (Kripke, 1974). The suppression induced by UV radiation is unique. Despite the limited ability of UV to penetrate tissue (Evertt et al., 1966), the suppression seen following exposure to UV radiation is systemic. For example, after a single exposure to UV radiation, mice are unable to generate a delayed-type hypersensitivity reaction to antigens injected subcutaneously at a distant non-irradiated site (Ullrich et al., 1986a; Ullrich, 1986b; Molendijk et al., 1987). The suppression is specific for the injected antigen, and associated with the appearance of splenic antigen-specific suppressor T lymphocytes (Ullrich et al., 1988). Although it is not entirely clear how UV-irradiation of the skin can result in the induction of systemic immunosuppression, most of the evidence to date supports the concept that UV-induced soluble suppressive factors are involved. Indeed a wide variety of soluble factors have been implicated in the induction of systemic suppression following UV exposure, including cis-urocanic acid (De Fabo et al., 1983), contra-IL-1 (Schwarz et al., 1987), IL-1, (Robertson et al., 1987) prostaglandins (Chung, H. T. et al., 1986), serum factors (Swartz, R. P, 1984; Harriott-Smith, T. G. et al., 1988), and factors isolated from UV-irradiated keratinocytes (Schwarz, T. A. et al., 1986; Kim, T. Y. et al., 1990; Ullrich, S. E. et al., 1990).
Furthermore, the suppression observed following UV exposure is unique in that the immunosuppressive effect is highly selective. Although cell mediated immune reactions such as the rejection of UV-induced tumor cells, delayed-type hypersensitivity (DTH) and contact hypersensitivity (CHS) are suppressed in UV-irradiated mice, most other immune reactions, especially antibody production, are normal (Spellman, C. W. et al., 1977; Norbury, K. C. et al., 1977). Similarly, injecting supernatants from UV-irradiated keratinocyte cultures only suppresses the induction of cellular immune reactions; antibody production is normal in factor-injected mice (Kim, T. Y. et al., 1990).
The present inventors and others have provided evidence for the role of keratinocyte-derived cytokines in the induction of suppression following UV exposure. (Kim, T. Y. et al., 1990; Ullrich, S. E. et al., 1990; Luger, T. A. et al., 1989; Aubin, F. et al., 1991). The immunosuppression seen following total-body UV exposure and the suppression observed after injecting supernatants from UV-irradiated keratinocytes are selective in nature. Although cellular immune reactions such as DTH are suppressed, antibody formation is normal. (Kim, T. Y. et al., 1990; Spellman, C. W. et al., 1977; Norbury, K. C. et al., 1977). Two of the major immunologic defects associated with UV-induced systemic suppression are the suppression of delayed-in-time hypersensitivity reactions and depressed antigen-presenting cell capability (Kripke, M. L. 1984).
Another prominent suppressive cytokine implicated in the suppression of CHS following UV exposure in vivo is TNF-.alpha.. (Yoshikawa, T. et al., 1990). Although others have reported that keratinocytes release TNF-.alpha. after exposure to UV radiation, (Kock, A. et al., 1990) the present inventors were unable by Western analysis to find TNF-.alpha. in the keratinocyte supernatant of the present invention nor did treatment of the supernatant with anti-TNF-.alpha. antibody neutralize the suppressive activity. However, although these observations demonstrate that there is no TNF-.alpha. in this suppressive supernatant they do not rule out a role for TNF-.alpha. in the induction of suppression following UV exposure. Ansel et al. (1990), found that a number of agents are capable of activating keratinocytes to release cytokines, including the cytokines themselves. For example, IL-1 MRNA expression was up-regulated by incubating the keratinocytes in IL-1, TNF-.alpha., or granulocyte-macrophage colony stimulating factor (GM-CSF). Similarly, GM-CSF mRNA expression was up-regulated after incubation with IL-2, TNF-.alpha., or GM-CSF. It is possible that a similar situation occurs with keratinocyte-derived suppressor cytokines.
The induction of suppression by keratinocyte-derived suppressive cytokines and the selective nature of the immunosuppression generated following the injection of supernatants from UV-irradiated keratinocytes led the present inventors to focus attention on a suppressive cytokine, IL-10. Studies by Mosmann and colleagues demonstrated that CD4+T lymphocytes can be divided into two subclasses based on the pattern of cytokines released after antigenic stimulation. Th1 cells secrete IL-2, IFN-.gamma. and lymphotoxin, whereas Th2 cells produce IL-4, IL-5, IL-6 and IL-10 following antigenic stimulation. For the most part, Th1 cells, because of the cytokines they release, are more active in providing help for cellular immune reactions, whereas Th2 cells are much more efficient at providing help for humoral immune reactions (Florentino, D. F. et al., 1989). Moreover, there appears to be a cross-regulation between these two subsets of helper cells during an immune response. IFN-.gamma. production by Th1 cells prevents the proliferation of Th2 cells, thus limiting humoral immune reactions, and IL-10 secreted by Th2 cells interferes with cytokine production by Th1 cells, thus limiting cellular immune reactions. This cross-regulation by Th1 and Th2 cells may help to explain the observation that antibody production and DTH are often mutually exclusive (Parish, C. R., 1972). IL-10 has been implicated in the suppression of DTH and inhibits antigen-presenting cell activity. (Florentino, D. F. et al., 1991; Mosmann, T. R., 1991).
Other keratinocyte and epidermal-derived soluble suppressive factors have also been implicated in the induction of immunosuppression following UV exposure (Ullrich, S. E., 1991). Two of the hallmarks of the systemic suppression induced by UV radiation are the suppression of DTH and CHS and a systemic depression of antigen-presenting cell capability. Of the various suppressive factors that have been implicated in the induction of systemic suppression by UV radiation, only a few have been shown to inhibit both delayed-in-time-hypersensitivity reactions and antigen-presenting cell function. Schwarz and colleagues have described factors from UV-irradiated keratinocytes that suppress CHS (contra-CHS) and IL-1-induced thymocyte activation (contra-IL-1). (Schwarz et al., 1987; Schwarz, T. A. et al., 1986). Contra-IL-1 has been found in the serum of UV-irradiated human volunteers and is associated with depressed antigen-presenting cell capability. (Krutmann, J. et al., 1990). UV exposure also promotes the conversion of trans-urocanic acid to the cis-isomer, and cis-urocanic acid suppresses both DTH reactions and systemic antigen-presenting cell activity. (Ross, J. A. et al., 1988; Noonan, F. P. et al., 1988). The present invention demonstrates that keratinocyte-derived IL-10 suppresses the induction of DTH. The present inventors have shown previously that splenic adherent cells isolated from mice injected with supernatants from UV-irradiated keratinocytes were ineffective at presenting haptens for a DTH reaction. (Ullrich, S. E., 1991). Furthermore, injecting supernatants from UV-irradiated keratinocytes into mice suppresses host resistance to Mycobacterium bovis BCG by suppressing DTH and interfering with bacterial clearance. (Jeevan, A. et al., 1992). This suppression of antigen-presenting cell activity and inhibition of microbial killing is consistent with the reported functions of IL-10. (Florentino, D. F. et al., 1991; Gazzinelli, R. T. et al., 1992).
The release of IL-10 by UV-irradiated keratinocytes may also explain the selective suppression (i.e., inhibition of cell-mediated immune reactions but not of antibody production) observed following UV exposure or the injection of supernatants from UV-irradiated keratinocytes. Therefore, the purpose of the present study was to determine whether IL-10 is released by keratinocytes following UV exposure. Furthermore, the present inventors also examined the ability of neutralizing monoclonal antibodies against IL-10 to inhibit the induction of systemic suppression observed following the injection of supernatants from UV-irradiated keratinocytes into mice or following total-body UV exposure.
The study of the systemic suppression of the immune system by UV-radiation is important for a number of reasons. First, an association between immunosuppression and the development of primary skin cancers in mice has been demonstrated Fischer, 1982!. Insight into the mechanism by which UV-radiation suppresses the immune response may be helpful in providing new approaches for the treatment and/or prevention of skin cancer. Second, the systemic immunologic alterations caused by UV-radiation, especially the suppression of DTH, may be a predisposing factor for an increased incidence of infectious diseases. This coupled with a decrease in the atmospheric ozone layer suggests that UV-induced immunosuppression may adversely affect the health of wide segments of the population. Finally, the immunosuppression induced by UV-radiation may have therapeutic applications, e.g., in the suppression of allograft rejection.
The present inventor has demonstrated that at least two factors are involved in UV induced CHS and DTH suppression, each being released by cells after irradiation with different wavelengths of UV-radiation. The present inventor has determined that supernatant from epidermal cells exposed to long-wave UV radiation, UVA, (320-400 nm) would suppress CHS but not DTH. On the other hand, supernatants from short-wave UV-radiation, UVB, (280-320 nm) would suppress DTH but not CHS. This result shows that two different immunosuppressive factors are released by UV-irradiated cells. The first immunosuppressive is released on exposure to UVB and suppresses DTH and the second is released on exposure to UVA and suppresses CHS. Therefore, by using a pre-determined wavelength of ultraviolet radiation (UVR), e.g., UVA or UVB, the immune response of a mammal can be selectively suppressed.
Typically, to overcome the immunological rejection of transplanted tissue (allografts), immunosuppressive drugs are used. One serious side effect of many of these agents, however, is the pan-immunosuppression that is produced. In addition to the suppression of allograft rejection, all other immune responses, such as those involved in the protection of the host from viral and bacterial pathogens, are also suppressed. As a result the immunosuppressed patient is susceptible to a variety of opportunistic infections. Accordingly, a method of suppressing only the immune response to the allografted tissue while leaving other immunological functions intact would be highly advantageous.
It has been reported that direct UV-irradiation (UVR) of the allograft can result in prolonged survival (Lau et al., 1983; Lau et al., 1984), of the allograft. The mechanism suggested there is an alteration of the antigenic composition of the grafted tissue by the UVR, thus rendering the allograft nonantigenic. In the present invention, however, an alternative approach of rendering the recipient tolerant to the allograft is taken.